Drosophila Genome Engineering

CRISPR/Cas9, TALEN, ZFN system are three powerful genome engineering tools that are composed of
programmable, sequence-specific DNA-binding modules associated with a nonspecific DNA nuclease,
which introduce DSB (DNA double-strand breaks) that stimulate NHEJ (non-homologous end joining) or
HR (homologous recombination) repairing mechanisms at specific genomic locations. In the application
of these technology in Drosophila, random indels can be introduced into DSB via NHEJ.

Currently, CRISPR/Cas9 has grown in popularity for Drosophila genome editing.

Drosophila CRISPR/Cas9 System

CRISPR/Cas system (clustered regularly interspaced palindromic repeats/CRISPR associated) is a component of bacteria/archaea innate immunity system. Type II CRISPR/Cas9 system uses ~20 nt sequence of crRNA to bind its complementary sequence of exogenous DNA. Subsequently with the aid of trans-acting RNA (trancrRNA), Cas9 endonuclease is recruited to the sequence and cut off the DNA at both strands.

In the application of CRISPR/Cas9 system in Drosophila genome editing, the two RNAs that play key roles can be synthesized into a single chimeric RNA (chiRNA or gRNA). By editing the 20 nt targeting sequence of gRNA, the system introduces DSB on the target sequence followed by an NGG or NAG protospacer adjacent motif (PAM) required for Cas9 cleavage.

For creating stable attP-gRNA-expressing lines, you may actually choose any of the vermillion+ based attP lines from these lists. We may setup the crosses to the y-v- line and screen for v+. You may get the same v+ G2 transformants as using the v- based attP lines. This is just regular Plan H or Plan I with extra v+ screening. You may also ask us to keep the resulting gRNA lines and have us cross to any of the Cas9 stocks and inject their F1s (additional Plan R) later.